RASSF1 promotes cardiomyocyte apoptosis after acute myocardial infarction and is regulated by miR-125b

Cardiomyocyte apoptosis is a common pathological injury in association with acute myocardial infarction (AMI). In the current study, the relationship between Ras-association domain family 1 (RASSF1) and cardiomyocyte apoptosis was investigated. RASSF1 was significantly over expressed in infarcted myocardial tissues as well as in cardiomyocytes induced by hypoxia. Inhibition of RASSF1 expression alleviated cardiomyocytes apoptosis induced by hypoxia in vitro and reduced cardiomyocytes apoptosis after AMI in vivo. RASSF1 expression was directly modulated by miR-125b, which was further confirmed by luciferase reporter assay. The current study verified that the miR-125b/RASSF1 axis was involved in cardiomyocytes apoptosis. To sum up, these results suggest that RASSF1 downregulation alleviated infarction-induced cardiomyocytes apoptosis and was regulated by miR-125b.     

SR-A deficiency reduces myocardial ischemia/reperfusion injury; involvement of increased microRNA-125b expression in macrophages

The macrophage scavenger receptor class A (SR-A) participates in the innate immune and inflammatory responses. This study examined the role of macrophage SR-A in myocardial ischemia/reperfusion (I/R) injury and hypoxia/reoxygenation (H/R)-induced cell damage. SR-A^?/? and WT mice were subjected to ischemia (45 min) followed by reperfusion for up to 7 days. SR-A^?/? mice showed smaller myocardial infarct size and better cardiac function than did WT I/R mice. SR-A deficiency attenuated I/R-induced myocardial apoptosis by preventing p53-mediated Bak-1 apoptotic signaling. The levels of microRNA-125b in SR-A^?/? heart were significantly greater than in WT myocardium. SR-A is predominantly expressed on macrophages. To investigate the role of SR-A macrophages in H/R-induced injury, we isolated peritoneal macrophages from SR-A deficient (SR-A^?/?) and wild type (WT) mice. Macrophages were subjected to hypoxia followed by reoxygenation. H/R markedly increased NF-κB binding activity as well as KC and MCP-1 production in WT macrophages but not in SR-A^?/? macrophages. H/R induced caspase-3/7 and -8 activities and cell death in WT macrophages, but not in SR-A^?/? macrophages. The levels of miR-125b in SR-A^?/? macrophages were significantly higher than in WT macrophages. Transfection of WT macrophages with miR-125b mimics attenuated H/R-induced caspase-3/7 and -8 activities and H/R-decreased viability, and prevented H/R-increased p-53, Bak-1 and Bax expression. The data suggest that SR-A deficiency attenuates myocardial I/R injury by targeting p53-mediated apoptotic signaling. SR-A^?/? macrophages contain high levels of miR-125b which may play a role in the protective effect of SR-A deficiency on myocardial I/R injury and H/R-induced cell damage.     

Endogenous estrogen mediates a higher threshold for endotoxin-induced myocardial protection in females

Myocardial endotoxin tolerance may be induced in both males and females; however, it remains unknown whether there are mechanistic and threshold differences between the sexes. We hypothesized that endogenous estrogen mediates a higher threshold for endotoxin (ETX)-induced protection in females. Adult proestrus and ovariectomized (OVX) female rats were preconditioned (PC) with intraperitoneal injections of 125 (PC+125) or 500 (PC+500) microg/kg Salmonella typhimurium LPS (ETX) or normal saline (PC-). Twenty-four hours later, injury dose ETX (500 microg/kg) was injected. After 6 h, myocardial function was measured via Langendorff. p38 MAPK and JNK activation and TNF-alpha, IL-1, and IL-6 expression were evaluated. ETX injury significantly decreased left ventricular developed pressure in PC- groups vs. controls. PC+500 regimen protected against ETX injury, resulting in normal cardiac function. PC+125 regimen protected OVX but not proestrus females, which had diminished myocardial function. Activated JNK and TNF-alpha increased in PC- but were diminished in PC+500 animals. Importantly, activated JNK and TNF increased in PC+125 proestrus females, whereas PC+125 OVX females displayed decreases in these molecules. There were no differences in p38 MAPK activation or expression of IL-1 or IL-6. These results demonstrate that proestrus females require a higher stimulus (PC+500) to achieve myocardial protection against ETX injury. Removal of endogenous estrogen (OVX) lowered the preconditioning threshold (PC+125), resulting in protection after lesser injury. Additionally, myocardial JNK and TNF expression was decreased in OVX PC+125 females, which correlated with myocardial function differences. Therefore, we conclude that endogenous estrogen mediates a higher threshold for ETX tolerance in female myocardium.     

Myocardial meta-[125l]iodobenzylguanidine uptake in awake genetically hypertensive rats at different ages: an autoradiographic study.

The purpose of this work was to evaluate changes in myocardial meta-[125I]iodobenzylguanidine ([125I]MIBG) uptake and distribution with age in awake spontaneously hypertensive rats (SHR) with respect to Wistar-Kyoto (WKY) rats. Rats were randomly divided into two groups, one for measuring myocardial [125I]MIBG uptake and distribution 4 h after its injection and the second for evaluating myocardial catecholamine concentrations. Mean arterial blood pressure, cardiac hypertrophy index (heart/body weight ratio), and heart rate were significantly higher with increasing age in SHR compared with matched WKY rats. Myocardial catecholamine concentrations and turnover did not differ between the two strains and were significantly decreased with increasing age. Myocardial [125I]MIBG uptake determined by gamma counting was similar in WKY rats and SHR and did not vary significantly with age when expressed as uptake density. However, in both strains of rats, [125I]MIBG uptake determined by autoradiography was significantly greater at the base of the heart than at the apex and midventricular levels, and the uptake values of young rats were significantly higher than those of older rats. In 21-week-old WKY rats and SHR, the highest [125I]MIBG uptake values were found in the right ventricle. Thus, quantitative autoradiography allowed detection of significant changes in myocardial [125I]MIBG uptake and showed its heterogeneous distribution in the rat heart.     

过表达微小RNA miR-125b对人胚胎干细胞增殖的影响

目的:通过在人胚胎干细胞(hESC)中有效转染微小RNA miR-125b的真核表达载体,研究过表达miR-125b对hESC增殖的影响。方法:将在无饲养层上培养至第3 d,克隆融合达70%的hESC用Accutase酶消化为单细胞,然后用LipofectAMINE2000对hESC单细胞转染pHRS-1cla-miR125b-CMV-EGFP载体及其对照pHRS-1cla-CMV-EGFP载体,通过实时定量PCR对转染后细胞中成熟miR-125b的表达进行检测;进一步进行细胞计数和克隆计数,对miR-125b表达上调的hESC的增殖情况进行分析。结果:实时定量PCR检测结果表明,细胞转染后72 h,miR-125b的表达上调1.45倍,说明hESC转染成功;克隆计数及细胞计数结果显示过表达miR-125b的hESC增殖受到明显抑制(P<001)。结论:转染miR-125b真核表达载体的hESC能够上调成熟miR-125b的表达,hESC中miR-125b的表达上调能明显抑制hESC的增殖。     

MicroRNA-125b protects liver from ischemia/reperfusion injury via inhibiting TRAF6 and NF-κB pathway

MicroRNA-125b (miR-125b), which was previously proved to be a potential immunomodulator in various disease, attenuated mouse hepatic ischemia/reperfusion (I/R) injury in this study. miR-125b was decreased in RAW 264.7 cells exposed to hypoxia/reoxygenation (H/R). The expression of IL-1β, IL-6 and TNF-α in both serum and supernate were reduced in miR-125b over-expression groups. The hepatic histopathological changes were reduced in miR-125b agomir groups. In the miR-125b antagomir groups, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly elevated compared with negative control (NC) groups. The protein expression of TNF receptor-associated factor 6 (TRAF6), IL-1β and the phosphorylation of p65 (p-p65) were suppressed by the up-regulation of miR-125b. Furthermore, the nuclear translocation of p-p65, measured by immunofluorescence, was enhanced by the miR-125b inhibitors. In conclusion, our study indicates that miR-125b protects liver from hepatic I/R injury via inhibiting TRAF6 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signal pathway.     

miRNA-125b在膀胱癌中的表达和意义

目的:探讨膀胱癌组织中miR-125b的表达在膀胱癌发生发展中的作用及其临床意义。方法:采用应用茎环RT-PCR的方法检测66例膀胱尿路上皮癌组织标本的miR-125b的表达,另有16例正常膀胱黏膜组织作对照,并结合临床病理资料进行统计学分析。结果:miR-125b在膀胱癌组织中的表达显著高于非肿瘤的正常膀胱黏膜组织(p0.05),miR-125b的水平还与表达水平与膀胱癌的组织学分级、肿瘤转移、术后复发均明显相关性(P0.05)。结论:miR-125b在膀胱癌组织中表达量升高,且与组织学分级、肿瘤转移、术后复发相关,可能作为膀胱癌诊断和预后指标。     

Modulation of myocardial alpha 1- but not beta-adrenoceptors after 90-day tail-suspension

We have previously demonstrated that prolonged simulated microgravity (tail-suspension) leads to cardiac alterations with increased resting heart rate, myocardial degradation changes and attenuated myocardial contractility. The present study investigated the potential role of adrenoceptor mechanisms underlying them. Changes of myocardial alpha 1-adrenoceptor (alpha 1-AR) and beta 1-adrenoceptor (beta-AR) in 90-day tail-suspended rats was investigated by the method of radioligand binding assay and application of Scatchard's method. The results showed significantly decreased quantity of specific binding of 125I-BE[2-beta-(4-hydroxy-3-[125I]indophenyl)-ethylaminomethyltetralone] to alpha 1-AR present in membrane derived from ventricular myocardium of the suspended animals, despite the affinity of the alpha 1-AR to 125I-Be was unchanged. But neither the quantity nor the affinity of beta-AR binding to 125I-Pindolol was significantly altered. In addition, the spontaneously beating rate of isolated right atria from tail-suspended animals showed little change in sensitivity and reactivity to the stimulations of graded phenylephrine (alpha-agonist, measured in the presence of beta-antagonist propranolol) and isoproterenol (beta-agonist), compared with the control rats. There were also no obvious differences of the effects of the isoproterenol on the contractility of isolated left ventricular papillary muscles between the two groups. Since myocardial alpha 1-AR mediated-effects include production of cardiac hypertrophy and enhancement of myocardial glucose uptake and glycolysis, the down-regulation of the alpha 1-AR may be a contributor to the cardiac cellular accumulation and the myocardial degradation changes as found in our tail-suspended rats. The data from this study also suggest that the myocardial beta-adrenoceptors are not affected by the prolonged tail-suspension.     

MiR-125b regulates the proliferation and metastasis of triple negative breast cancer cells via the Wnt/β-catenin pathway and EMT

Background/aim: MiR-125b plays an important role in breast cancer. The current study was to explore the expression and function of miR-125b in triple negative breast cancer cells. Materials and methods: The expression of miR-125b in human TNBC samples and cell lines were examined by qRT-PCR. MTT, scratch assays and transwell assays were utilized to observe the proliferation, migration and invasion ability. MiR-125b’s target gene and downstream signaling pathways were investigated by Luciferase Reporter Assays, qRT-PCR, immunofluorescence assays and western bolt. Results: MiR-125b was highly expressed in human TNBC tissues and cell lines. Inhibiting miR-125b expression suppressed the proliferation, cell migration and invasion. The three-prime untranslated region (3´-UTR) of adenomatous polyposis coli (APC) mRNA contains miR-125b binding sites, and inhibiting miR-125b expression suppressed the activity of the intracellular Wnt/β-catenin pathways and EMT. Conclusion: Inhibiting miR-125b regulates the Wnt/β-catenin pathway and EMT to suppress the proliferation and migration of MDA-MB-468 TNBC cells.     

大鼠心肌梗塞后心室重构中miRNA 的差异表达

目的：筛选大鼠急性心梗后的心室重构过程中差异表达的微小RNA(microRNA, miRNA),为miRNA 调控心室重构的机制 研究提供靶标。方法：20 只成年雄性Wistar 大鼠,分组如下：心肌梗死组(MI组,n=10)和假手术组(Sham 组,n=10)。通过结扎大鼠 左冠状动脉前降支构建急性心梗模型建模。4 周后对大鼠进行超声心动图检查和梗死边缘区心肌HE 染色观察心室重构程度。利 用miRNA芯片对心梗边缘区的miRNA进行差异表达检测,采用实时定量PCR验证芯片结果的可靠性。结果：心脏超声显示MI 组大鼠左室重构明显,心梗边缘区心肌HE染色可见细胞间质大量胶原纤维沉积。miRNA 芯片结果显示15 个miRNA在心梗4 周后呈差异表达,其中11 个miRNA(miR-21、miR-23a、miR-125b、miR-132、miR-146b、miR-181b、miR-199a、miR-320、miR-324、 miR-328 和miR-499)表达上调,4 个miRNA(miR-29、miR-30c、miR-133a 和miR-208)表达下调。实时定量PCR 验证结果与芯片结 果一致。结论：这些差异表达的miRNA 可能与心梗后心室重构相关,进一步深入研究特定miRNA 的调控机制有望为基因治疗提 供新靶点。     

过表达微小RNA miR-125b抑制肝癌细胞上皮-间质转换及促进细胞凋亡

微小RNA-125b（miR-125b）在许多恶性肿瘤的增殖、分化和凋亡等过程中具有很重要的作用,但miR-125b是否涉及肝癌的上皮 间质转换过程（EMT）还有待进一步研究。本研究通过构建过表达miR-125b的肝癌稳转细胞株,初步检测miR-125b对于肝癌的EMT过程和相关的TGF-β信号通路的影响,以及对于肝癌细胞凋亡的影响。以慢病毒载体pHRS-1cla EGFP 构建过表达miR-125b的载体质粒(pHRS-1cla-miR125b-CMV-EGFP),并对上述载体进行NheⅠ、XbaⅠ双酶切和测序鉴定,鉴定正确后,在293T细胞中进行慢病毒包装,浓缩病毒后,对MHCC97-H进行慢病毒感染并采用流式分选GFP阳性的细胞。实时定量PCR检测表明肝癌细胞稳转株MHCC97-H-PHRS-miR-125b-EGFP的miR-125b表达量是空载体转染组的6倍。Western印迹检测发现,与空载体对照组相比,MHCC97-H-PHRS-miR-125b-EGFP细胞中间质细胞标志α-SMA表达显著下调,上皮细胞标志E-cadherin表达显著上调,同样的,用Western印迹检测也发现MHCC97-H-PHRS-miR-125b-EGFP细胞中TGF-β信号通路关键下游分子Smad2和Smad4的表达显著下调,细胞凋亡检测结果表明,与对照组相比,过表达miR-125b的稳转株凋亡率增加到19.66%,加入TGF-β1后,过表达miR-125b的稳转株凋亡率进一步增加到74.7%。同样的,在体内治疗实验中,我们采用商品化的体内核酸转染试剂,在皮下肿瘤组织中过表达miR-125b mimics,结果表明miR-125b的过表达与肿瘤组织的凋亡成正相关性（r=0.83463,P < 0.01）,且免疫组化结果也表明,miR-125b过表达后,E-cadherin表达显著上调,α-SMA及Smad2和Smad4的表达显著下调。上述结果表明,我们成功构建了过表达miR-125b的肝癌细胞稳转株,并成功建立了肿瘤组织中过表达miR-125b mimics的动物模型,在体内外均观察到过表达miR-125b后对肝癌细胞EMT过程的抑制作用和对细胞凋亡的促进作用。相关研究结果加深了我们对miR-125b在肝癌中抑制肝癌发展作用机制的理解,及其作为潜在的治疗肝癌的新靶点的重要性。     

MicroRNA-125b potentiates macrophage activation

MicroRNA (miR)-125b expression is modulated in macrophages in response to stimulatory cues. In this study, we report a functional role of miR-125b in macrophages. We found that miR-125b is enriched in macrophages compared with lymphoid cells and whole immune tissues. Enforced expression of miR-125b drives macrophages to adapt an activated morphology that is accompanied by increased costimulatory factor expression and elevated responsiveness to IFN-γ, whereas anti-miR-125b treatment decreases CD80 surface expression. To determine whether these alterations in cell signaling, gene expression, and morphology have functional consequences, we examined the ability of macrophages with enhanced miR-125b expression to present Ags and found that they better stimulate T cell activation than control macrophages. Further indicating increased function, these macrophages were more effective at killing EL4 tumor cells in vitro and in vivo. Moreover, miR-125b repressed IFN regulatory factor 4 (IRF4), and IRF4 knockdown in macrophages mimicked the miR-125b overexpression phenotype. In summary, our evidence suggests that miR-125b is at least partly responsible for generating the activated nature of macrophages, at least partially by reducing IRF4 levels, and potentiates the functional role of macrophages in inducing immune responses.     

miR-125b targets ARID3B in breast cancer cells

Mounting evidence suggests involvement of deregulated microRNA (miRNA) expression during the complex events of tumorigenesis. Among such deregulated miRNAs in cancer, miR-125b expression is reported to be consistently low in breast cancers. In this study, we screened a panel of breast cancer cell lines (BCCLs) for miR-125b expression and detected decreased expression in 14 of 19 BCCLs. Due to the heterogeneity of breast cancers, MCF7 cells were chosen as a model system for ERBB2 independent breast cancers to restore miR-125b expression (MCF7-125b) to investigate the phenotypical and related functional changes. Earlier, miR-125b was shown to regulate cell motility by targeting ERBB2 in ERBB2 overexpressing breast cancer cells. Here we showed decreased motility and migration in miR-125b expressing MCF7 cells, independent of ERBB2. MCF7-125b cells demonstrated profoundly decreased cytoplasmic protrusions detected by phalloidin staining of filamentous actin along with decreased motility and migration behaviors detected by in vitro wound closure and transwell migration assays compared to empty vector transfected cells (MCF7-EV). Among possible numerous targets of miR-125b, we showed ARID3B (AT-rich interactive domain 3B) to be a novel target with roles in cell motility in breast cancer cells. When ARID3B was transiently silenced, the decreased cell migration was also observed. In light of these findings, miR-125b continues to emerge as an interesting regulator of cancer related phenotypes.     

微小RNA miR-125b真核表达载体的构建及其对细胞增殖的影响

目的:构建微小RNA125b(miR-125b)真核表达载体,研究其过表达后对细胞增殖的影响。方法:以pcDNA3.1(-)-myc-his载体为模板,PCR扩增CMV启动子,克隆入pHRS-1cla-EGFP慢病毒载体,构建pHRS-1cla-CMV-EGFP载体;以从人全血中提取的基因组DNA为模板,PCR扩增pri-miR-125b序列,将其克隆到pHRS-1cla-CMV-EGFP载体中,构建pHRS-1cla-miR125b-CMV-EGFP慢病毒表达载体;将pHRS-1cla-miR125b-CMV-EGFP表达载体瞬时转染入293FT细胞,用实时定量PCR技术对miR-125b在转录水平的表达进行检测,用MTT及Brdu法检测miR-125b过表达后对293FT细胞增殖的影响。结果:构建的pHRS-1cla-miR125b-CMV-EGFP真核表达载体经质粒酶切和测序鉴定正确,转染细胞后72h经实时定量PCR检测,成熟miR-125b表达上调约750倍(P0.01),说明其能有效高表达,MTT及Brdu法检测显示细胞增殖受到明显抑制(P0.01)。结论:构建了pHRS-1cla-miR-125b-CMV-EGFP慢病毒真核表达载体,转染293FT细胞后能高效表达成熟miR-125b,同时证明过表达miR-125b能使细胞的增殖受到非常明显的抑制。     

MicroRNA-125b induces metastasis by targeting STARD13 in MCF-7 and MDA-MB-231 breast cancer cells

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression by targeting mRNAs to trigger either translation repression or mRNA degradation. miR-125b is down-regulated in human breast cancer cells compared with the normal ones except highly metastatic tumor cells MDA-MB-231. However, few functional studies were designed to investigate metastatic potential of miR-125b. In this study, the effects of miR-125b on metastasis in human breast cancer cells were studied, and the targets of miR-125b were also explored. Transwell migration assay, cell wound healing assay, adhesion assay and nude mice model of metastasis were utilized to investigate the effects of miR-125b on metastasis potential in vitro and in vivo. In addition, it was implied STARD13 (DLC2) was a direct target of miR-125b by Target-Scan analysis, luciferase reporter assay and western blot. Furthermore, activation of STARD13 was identified responsible for metastasis induced by miR-125b through a siRNA targeting STARD13. qRT-PCR, immunofluorescent assay and western blot was used to observe the variation of Vimentin and α-SMA in breast cancer cells. In summary, our study provided new insights into the function of miR-125b during the metastasis of breat cancer cells and also suggested the role of miR-125b in pro-metastasis by targeting STARD13.